In recent years, a myriad of reports have appeared on the attempted specific targeting of tumor cells with monoclonal antibody-drug conjugates {(Sela et al, in Immunoconjugates, pp. 189-216 (C. Vogel, ed. 1987); Ghose et al, in Targeted Drugs, pp. 1-22 (E. Goldberg, ed. 1983); Diener et al, in Antibody Mediated Delivery Systems, pp. 1-23 (J. Rodwell, ed. 1988); Pietersz et al, in Antibody Mediated Delivery Systems, pp. 25-53 (J. Rodwell, ed. 1988); Bumol et al, in Antibody Mediated Delivery Systems, pp. 55-79 (J. Rodwell, ed. 1988)}. Cytotoxic drugs such as methotrexate, daunorubicin, doxorubicin, vincristine, vinblastine, melphalan, mitomycin C, chlorambucil, and maytansinoids have been conjugated to a variety of murine monoclonal antibodies. In some cases, the drug molecules were linked to the antibody molecules through an intermediary carrier molecule such as serum albumin {Garnett et al, 46 Cancer Res. 2407-2412 (1986); Ohkawa et al, 23 Cancer Immunol. Immunother. 81-86 (1986); Endo et al, 47 Cancer Res. 1076-1080 (1980)}, dextran {Hurwitz et al, 2 Appl. Biochem. 25-35 (1980); Manabi et al, 34 Biochem. Pharmacol. 289-291 (1985); Dillman et al, 46 Cancer Res. 4886-4891 (1986); Shoval et al, 85 Proc. Natl. Acad. Sci. U.S.A. 8276-8280 (1988)}, or polyglutamic acid {Tsukada et al, 73 J. Natl. Canc. Inst. 721-729 (1984); Kato et al, 27 J. Med. Chem. 1602-1607 (1984); Tsukada et al, 52 Br. J. Cancer 111-116 (1985)}.
A wide array of linker technologies have been employed for the preparation of such immunoconjugates and both cleavable and non-cleavable linkers have been investigated. In most cases, the full cytotoxic potential of the drugs could only be observed, however, if the drug molecules could be released from the conjugates in unmodified form at the target site.
One of the cleavable linkers that has been employed for the preparation of antibody-drug conjugates is an acid-labile linker based on cis-aconitic acid that takes advantage of the acidic environment of different intracellular compartments such as the endosomes encountered during receptor mediated endocytosis and the lysosomes. Shen and Ryser introduced this method for the preparation of conjugates of daunorubicin with macromolecular carriers {102 Biochem. Biophys. Res. Commun. 1048-1054 (1981)}. Yang and Reisfeld used the same technique to conjugate daunorubicin to an anti-melanoma antibody {80 J. Natl. Canc. Inst. 1154-1159 (1988)}. Dillman et al also used an acid-labile linker in a similar fashion to prepare conjugates of daunorubicin with an anti-T cell antibody {48 Cancer Res. 6097-6102 (1988)}.
An alternative approach, explored by Trouet et al, involved linking daunorubicin to an antibody via a peptide spacer arm {79 Proc. Natl. Acad. Sci. U.S.A. 626-629 (1982)}. This was done under the premise that free drug could be released from such a conjugate by the action of lysosomal peptidases.
In vitro cytotoxicity tests, however, have revealed that antibody-drug conjugates rarely achieved the same cytotoxic potency as the free unconjugated drugs. This suggested that mechanisms by which drug molecules are released from the antibodies are very inefficient. In the area of immunotoxins, conjugates formed via disulfide bridges between monoclonal antibodies and catalytically active protein toxins were shown to be more cytotoxic than conjugates containing other linkers. See, Lambert et al, 260 J. Biol. Chem. 12035-12041 (1985); Lambert et al, in Immunotoxins 175-209 (A. Frankel, ed. 1988); Ghetie et al, 48 Cancer Res. 2610-2617 (1988). This was attributed to the high intracellular concentration of glutathione contributing to the efficient cleavage of the disulfide bond between an antibody molecule and a toxin. Despite this, there are only a few reported examples of the use of disulfide bridges for the preparation of conjugates between drugs and macromolecules. Shen et al described the conversion of methotrexate into a mercaptoethylamide derivative followed by conjugation with poly-D-lysine via a disulfide bond {260 J. Biol. Chem. 10905-10908 (1985)}. A recent report described the preparation of a conjugate of the trisulfide containing toxic drug calicheamycin with an antibody {Menendez et al, Fourth International Conference on Monoclonal Antibody Immunoconjugates for Cancer, San Diego, Abstract 81 (1989)}.
One reason for the lack of disulfide linked antibody-drug conjugates is the unavailability of cytotoxic drugs possessing a sulfur atom containing moiety that can be readily used to link the drug to an antibody via a disulfide bridge. Furthermore, chemical modification of existing drugs is difficult without diminishing their cytotoxic potential.
Another major drawback with existing antibody-drug conjugates is their inability to deliver a sufficient concentration of drug to the target site because of the limited number of targeted antigens and the relatively moderate cytotoxicity of cancerostatic drugs like methotrexate, daunorubicin and vincristine. In order to achieve significant cytotoxicity, linkage of a large number of drug molecules either directly to the antibody or through a polymeric carrier molecule becomes necessary. However such heavily modified antibodies often display impaired binding to the target antigen and fast in vivo clearance from the blood stream.
CC-1065 is a potent antitumor-antibiotic isolated from cultures of Streptomyces zelensis and has been shown to be exceptionally cytotoxic in vitro.
The structure of CC-1065 (Compound 1, FIG. 1A) has been determined by X-ray crystallography {Martin, D. G. et al, 33 J. Antibiotics 902-903 (1980), and Chidester, C. G., et al, 103 J. Am. Chem. Soc. 7629-7635 (1981)}. The CC-1065 molecule consists of 3 substituted pyrroloindole moieties linked by amide bonds. The "A" subunit has a cyclopropyl ring containing the only asymmetric carbons in the molecule. While only the relative configuration of these carbons is available from X-ray data, the absolute configuration has been inferred as 3bR, 4as by using DNA as a chiral reagent {Hurley, L. H. et al, 226 Science 843-844 (1984)}. The "B" and "C" subunits are identical pyrroloindole moieties.
The cytotoxic potency of CC-1065 has been correlated with the alkylating activity and the DNA-binding or DNA-intercalating activity of CC-1065. The two activities reside in two separate parts of the molecule. The alkylating activity is contained in the CPI unit A (cyclopropapyrroloindole unit) and the DNA-binding in the two subunits B and C (see FIG. 1A).
CC-1065 is 100 to 1000-fold more cytotoxic than conventional cancer chemotherapeutic agents such as methotrexate, daunorubicin and vincristine {B. K. Bhuyan et al, 42 Cancer Res. 3532-3537 (1982)}. However, administration of CC-1065 to mice causes a delayed hepatotoxicity leading to death at 50 days after a single i.v. dose of 12.5 .mu.g/kg {V. L. Reynolds et al, XXXIX J. Antibiotics 319-334 (1986)}. The synthesis of some new analogues of CC-1065 (FIG. 1B and 1C) that retain the high in vitro cytotoxicity of the parent drug without causing delayed lethality in mice have been reported recently {M. A. Warpehoski et al, 31 J. Med. Chem. 590-603 (1988)}. Like CC-1065, these analogues are alkylating agents that bind to DNA in a covalent manner causing cell death. These compounds inhibit the growth of L1210 murine leukemia cells in vitro with IC.sub.50 values in the range of 1.times.10.sup.-10 to 1.times.10.sup.-11 M. Some of these compounds display in vivo efficacy against P388 leukemia in mice. The most effective analogue U-73975 (FIG. 1C) shows a 170% increase in life span over untreated controls at the optimal dose of 0.05 mg/kg given i.p. on days 1, 5, and 9. However, at this dose, only 1 out of 6 treated mice survived beyond 30 days. Therefore, these drugs have very little therapeutic value because of their high toxicity at concentrations necessary for therapeutic effects.
Accordingly, a need exists to improve the therapeutic efficacy of CC-1065 and its analogues such that the compounds retain cytotoxic activity but have decreased systemic toxicity.